Vaccines expressed in plants

ABSTRACT

The anti-viral vaccine of the present invention is produced in transgenic plants and then administered through standard vaccine introduction method or through the consumption of the edible portion of those plants. A DNA sequence encoding for the expression of a surface antigen of a viral pathogen is isolated and ligated to a promoter which can regulate the production of the surface antigen in a transgenic plant. This gene is then transferred to plant cells using a procedure that results in its integration into the plant genome, such as through the use of an Agrobacterium tumefaciens plasmid vector system. Preferably, the foreign gene is expressed in an portion of the plant that is edible by humans or animals. In a preferred procedure, the vaccine is administered through the consumption of the edible plant as food, preferably in the form of a fruit or vegetable juice.

This is a divisional of U.S. application Ser. No. 08/026,393 filed Mar.4, 1993 (now U.S. Pat. No. 5,612,487) and a continuation-in-part of PCTapplication PCT/US94/02332 filed Mar. 4, 1994 (designating the U.S.)which is a continuation-in-part of U.S. application Ser. No. 07/750,049filed Aug. 26, 1991 (abandoned). This application is also acontinuation-in-part of U.S. application Ser. No. 08/156,508 filed Nov.23, 1993 (now U.S. Pat. No. 5,484,719).

BACKGROUND OF THE INVENTION

This invention relates generally to vaccines and more particularly tothe production of oral vaccines in edible transgenic plants and theadministration of the oral vaccines such as through the consumption ofthe edible transgenic plants by humans and animals.

Diseases have been a plague on civilization for thousands of years,affecting not only man but animals. In economically advanced countriesof the world, diseases are 1) temporarily disabling; 2) permanentlydisabling or crippling; or 3) fatal. In the lesser developed countries,diseases tend to fall into the latter two categories, permanentlydisabling or crippling and fatal, due to many factors, including a lackof preventative immunization and curative medicine.

Vaccines are administered to humans and animals to induce their immunesystems to produce antibodies against viruses, bacteria, and other typesof pathogenic organisms. In the economically advanced countries of theworld, vaccines have brought many diseases under control. In particular,many viral diseases are now prevented due to the development ofimmunization programs. The virtual disappearance of smallpox, certainly,is an example of the effectiveness of a vaccine worldwide. But manyvaccines for such diseases as poliomyelitis, measles, mumps, rabies,foot and mouth, and hepatitis B are still too expensive for the lesserdeveloped countries to provide to their large human and animalpopulations. Lack of these preventative measures for animal populationscan worsen the human condition by creating food shortages.

The lesser developed countries do not have the monetary funds toimmunize their populations with currently available vaccines. There isnot only the cost of producing the vaccine but the further cost of theprofessional administration of the vaccine. Also, some vaccines requiremultiple doses to maintain immunity. Therefore, often, the countriesthat need the vaccines the most can afford them the least.

Underlying the development of any vaccine is the ability to grow thedisease causing agent in large quantities. At the present, vaccines areusually produced from killed or live attenuated pathogens. If thepathogen is a virus, large amounts of the virus must be grown in ananimal host or cultured animal cells. If a live attenuated virus isutilized, it must be clearly proven to lack virulence while retainingthe ability to establish infection and induce humoral and cellularimmunity. If a killed virus is utilized, the vaccine must demonstratethe capacity of surviving antigens to induce immunization. Additionally,surface antigens, the major viral particles which induce immunity, maybe isolated and administered to proffer immunity in lieu of utilizinglive attenuated or killed viruses.

Vaccine manufacturers often employ complex technology entailing highcosts for both the development and production of the vaccine.Concentration and purification of the vaccine is required, whether it ismade from the whole bacteria, virus, other pathogenic organism or asub-unit thereof. The high cost of purifying a vaccine in accordancewith Food and Drug Administration (FDA) regulations makes oral vaccinesprohibitively expensive to produce because they require ten to fiftytimes more than the regular quantity of vaccine per dose than a vaccinewhich is parenterally administered. Of all the viral vaccines beingproduced today only a few are being produced as oral vaccines.

According to FDA guidelines, efficacy of vaccines for humans must bedemonstrated in animals by antibody development and by resistance toinfection and disease upon challenge with the pathogen. When the safetyand immunogenicity levels are satisfactory, FDA clinical studies arethen conducted in humans. A small carefully controlled group ofvolunteers are enlisted from the general population to begin humantrials. This begins the long and expensive process of testing whichtakes years before it can be determined whether the vaccine can be givento the general population. If the trials are successful, the vaccine maythen be mass produced and sold to the public.

Even after these precautions are taken, problems can arise. With thekilled virus vaccines, there is always a chance that one of the liveviruses has survived and vaccination may lead to isolated cases of thedisease. Moreover, since both the killed and live attenuated types ofvirus vaccines are made from viruses grown in animal host cells, thevaccines are sometimes contaminated with cellular material from theanimal host which can cause adverse, sometimes fatal, reactions in thevaccine recipient. Legal liability of the vaccine manufacturer for thosewho are harmed by a rare adverse reaction to a new or improved vaccinenecessitates expensive insurance which ultimately adds to the cost ofthe vaccine.

Some vaccines have other disadvantages. Vaccines prepared from wholekilled virus generally stimulate the development of circulatingantibodies (IgM, IgG) thereby conferring a limited degree of immunitywhich usually requires boosting through the administration of additionaldoses of vaccine at specific time intervals. Live attenuated viralvaccines, while much more effective, have limited shelf-life and storageproblems requiring maintaining vaccine refrigeration during delivery tothe field.¹

Efforts today are being made to produce less expensive vaccines whichcan be administered in a less costly manner. Recombinants or mutants canbe produced that serve in place of live virus vaccines. The developmentof specific deletion mutants that alter the virus, but do not inactivateit, yield vaccines that can replicate but cannot revert to virulence.

Recombinant DNA techniques are being developed to insert the gene codingfor the immunizing protein of one virus into the genome of a second, avirulent virus type that can be administered as the vaccine. Recombinantvaccines may be prepared by means of a vector virus such as vaccinesvirus or by other methods of gene splicing. Vectors may include not onlya virulent viruses but bacteria as well. A live recombinant hepatitis Avaccine has been constructed using attenuated Salmonella typhimurium asthe delivery vector via oral administration.¹

Various a virulent viruses have been used as vectors. The gene forhepatitis B surface antigen (HMsAg) has been introduced into a genenon-essential for vaccines replication. The resulting recombinant virushas elicited an immune response to the hepatitis B virus in testanimals. Additionally, researchers have used attenuated bacterial cellsfor expressing hepatitis B antigen for oral immunization. Importantly,when whole cell attenuated Salmonella expressing recombinant hepatitisantigen were fed to mice, anti-viral T and B cell immune responses wereobserved. These responses were generated after a single oralimmunization with the bacterial cells resulting in high-titers of theantibody. See, e.g., "Expression of hepatitis B virus antigens inattenuated Salmonella for oral immunization," F. Schodel and H. Will,Res. Microbiol., 141:831-837 (1990). Others have had similar successwith oral administration routes for recombinant hepatitis antigens. See,e.g., M. D. Lubeck et al., "Immunogenicity and efficiacy testing inchimpanzees of an oral hepatitis B vaccine based on live recombinantadenovirus," Proc. Natl. Acad. Sci. 86:6763-6767 (1989); S. Kuriyama, etal., "Enhancing effects of oral adjuvants on anti-HBs responses inducedby hepatitis B vaccine," Clin. Exp. Immunol. 72:383-389 (1988).

Other virus vectors may possess large genomes, e.g. the herpesvirus. Theoral adenovirus vaccine has been modified so that it carries the HBsAgimmunizing gene of the hepatitis B virus. Chimeric polio virus vaccineshave been constructed of which the completely a virulent type 1 virusacts as a vector for the gene carrying the immunizing VP1 gene of type3.¹

Immunity to a pathogenic infection is based on the development of animmune response to specific antigens located on the surface of apathogenic organism. For enveloped viruses, the important antigens arethe surface glycoproteins. Glycosylation of viral surface glycoproteinsis not always essential for antigenicity.¹ Unglycosylated herpesvirusproteins synthesized in bacteria have been shown to produce neutralizingantibodies in test animals.¹ However, where recombinant antigens such asHBsAg are produced in organisms requiring complex fermentative processesand machinery, the costs and access can be prohibitive.

Viral genes which code for a specific surface antigen that producesimmunity in humans or animals, can be cloned into plasmids. The clonedDNA can then be expressed in prokaryotic or eukaryotic cells ifappropriately engineered constructions are used. The immunizing antigensof hepatitis B virus,² foot and mouth,³ rabies virus, herpes simplexvirus, and the influenza virus have been successfully synthesized inbacteria or yeast cells.¹

Animal and human subjects infected by a pathogen present an immuneresponse when overcoming the invading microorganism. They do so byinitiating at least one of three branches of the immune system mucosal,humoral or cellular. Mucosal immunity results from the production ofsecretory IgA antibodies in the secretions that bathe mucosal surfacesin the respiratory tract, the gastrointestinal tract, the genitourinarytact and the secretory glands. McGhee, J. R. et al. Annals NY Acad.Sci.409:409 (1983). Mucosal antibodies act to prevent colonization ofthe pathogen on mucosal surfaces thus establishing a first line ofdefense against invasion. The production of mucosal antibodies can beinitiated by either local immunization of the secretory gland or tissueor by presentation of the antigen to either the gut-associated lymphoidtissues (GALT; Peyer's Patches) or the bronchial-associated lymphoidtissue (BALT). Cebra, J. J. et al. Cold Spring Harbor Symp. Quant. Biol.41:210 (1976); Bienenstock, J. M., Adv. Exp. Med. Biol. 107:53 (1978);Weisz-Carrington, P. et al., J. Immunol. 123:1705 (1979); McCaughai, G.et al., Internal Rev. Physiol. 28:131 (1983). Humoral immunity, on theother hand, results from the production of IgG and IgM antibodies in theserun, precipitating phagocytosis of invading pathogens, neutralizationof viruses, or complement-mediated cytotoxicity against the pathogen.See, Hood et al. supra.

Others have noted that the induction of serum or mucosal antibodyresponses to orally administered antigens, however, may be problematic.Generally, such oral administration requires relatively large quantitiesof antigen since the amount of the antigen that is actually absorbed andcapable of eliciting an immune response is usually low. Thus, the amountof antigen required for oral administration generally far exceeds thatrequired for parenteral administration. de Aizpurua and Russell-Jones,J. Exp. Med. 167:440-451 (1988). However, it has been found that thesystemic and mucosal immune systems may be stimulated by feeding lowdoses of certain classes of proteins. In particular, this may beachieved with proteins which share the property of being able to bindspecifically to various glycolipids and glycoproteins located on thesurface of the cells on the mucosal membrane. Such proteins, called"mucosal immunogens" have been found to include viral antigens such asviral hemagglutinin. Moreover, dose-response experiments comparing oralwith intramuscular administration revealed that oral presentation ofmucosal immunogens was remarkably efficient in eliciting a serumantibody response to the extent that the response elicited by oralpresentation was only slightly lower than that elicited by intramuscularinjection of the mucosal immunogen. de Aizpurua and Russell-Jones,supra.

The hypothesis proposed by these workers that such mucosal immunogensshared a common ability to bind glycosylated surface proteins on themucosal membrane was at least partially confirmed by the inhibition ofmucosal uptake of these mucosal immunogens by certain high levels ofthree specific sugars (galactose, lactose or sorbitol). Other sugars,fructose (the principal sugar found in many plant fruits) mannose andmelibiose, did not inhibit mucosal immunogens from eliciting antibodies.de Aizpurua and Russell-Jones, supra. Others have found that certainsugars may, in fact, boost mucosal responses in the intestine. See,e.g., "Boosted Mucosal Immune Responsiveness in the Intestine byActively Transported Hexose," S. Zhang and G. A. Castro, Gastroenterol.,accepted for publication).

Recent advances in genetic engineering have provided the requisite toolsto transform plants to contain foreign genes. Plants that contain thetransgene in all cells can then be regenerated and can transfer thetransgene to their offspring in a Mendelian fashion.⁴ Bothmonocotyledenous and dicotyledenous plants have been stably transformed.For example, tobacco, potato and tomato plants are but a few of thedicotyledenous plants which have been transformed by cloning a genewhich encodes the expression of 5-enolpyruvyl-shikimate-3-phosphatesyntase.⁵

Plant transformation and regeneration in dicotyledons by Agrobacteriumtumefaciens (A. tumefaciens) is well documented. The application of theAgrobacterium tumefaciens system with the leaf disc transformationmethod⁶ permits efficient gene transfer, selection and regeneration.

Monocotyledons have also been found to be capable of genetictransformation by Agrobacterium tumefaciens as well as by other methodssuch as direct DNA uptake mediated by PEG (polyethylene glycol), orelectroporation. Successful transfer of foreign genes into corn⁷ andrice,⁸,9 as well as wheat and sorghum protoplasts has been demonstrated.Rice plants have been regenerated from untransformed and transformedprotoplasts. New methods such as microinjection and particle bombardmentmay offer simpler and even more efficient means of transformation andregeneration of monocotyledons.¹⁰

Attempts to produce tnansgenic plants expressing bacterial antigens ofEscherichia coli and of Streptococcus mutans have been made (Curtiss andIhnen, WO 90/0248, Mar. 22, 1990). However, until the work of thepresent inventors, no transgenic plants had been constructed expressingviral antigens such as HBsAg.⁷² In particular, until the work of thepresent inventors no such plants had been obtained which were capable ofexpressing viral antigens capable of eliciting an immune response as amucosal immunogen. Moreover, until the work reported above no suchplants had been obtained capable of producing particles which wereantigenically and physically similar to the commercially available HBsAgviral antigens derived from human serum or recombinant yeast. However,none of these references provided the possibility of testing trulyedible vaccines since all such studies were carried out in the classicaltobacco test systems which plant tissues are not routinely digested byman or animal.

Thus, while prior approaches to obtaining less expensive and moreaccessible vaccines have been attempted, there remains a need to providealternative sources of such vaccines for new antigens. Particularly,there remains need to provide alternative sources of vaccines which areincorporated by plants which are routinely included in human and animaldiets. For instance, while vaccines such as HBsAg have been producedusing antigen particles derived from human serum and recombinant yeastcells, both sources require greater expense and provide loweraccessibility to technically underdeveloped nations. Furthermore, whilecertain bacterial antigens may be expressed in transgenic plants, untilthe work of the present inventors it was unknown whether antigensassociated with human or animal viruses could be expressed in a formphysically and antigenically similar to antigens used in commercialvaccines derived from human serum or recombinant yeasts. Similarly,while it is now possible to produce such recombinant antigens in tobaccoplants by virtue of the present inventors work no such antigens havebeen produced in plants routinely included in human and animal diets. Inparticular, prior art approaches have filed to provide such commerciallyviable antigen from plants made to express transgenic hepatitis B viralantigens. Viral antigens, anti-viral vaccines and transgenic plantsexpressing the same as well as methods of making and using suchcompositions of matter are needed which provide inexpensive and highlyaccessible sources of such medicines in common diet plants of man andanimal.

SUMMARY OF THE INVENTION

Recombinant viral antigens, anti-viral vaccines and transgenic plantsexpressing the same are provided by the present invention. Thesecompositions of matter are demonstrated by the present invention to bemade and used by the methods of the invention in a manner which ispotentially less expensive as well as more accessible to lowertechnological societies which rely chiefly on agricultural methods toprovide essential raw materials.

More particularly, the present invention overcomes at least some of thedisadvantages of the prior art by providing antigens produced in edibletransgenic plants which antigens are antigenically and physicallysimilar to those currently used in the manufacture of anti-viralvaccines derived from human serum or recombinant yeasts. In a preferredembodiment, these compositions of matter and methods provide transgenicplants, recombinant viral antigens and anti-viral vaccines related tothe causative agent of human and animal viral diseases. The diseases ofparticular interest are those diseases in which the virus possesses anantigen capable, in at least the native state of the virus, of elicitingimmune responses, particularly mucosal immune responses. In anembodiment of preference, the pathogen from which the antigen is derivedis the hepatitis pathogen, and in plants which are routinely included inhuman and animal diets.

In one embodiment, the compositions of matter and methods of theinvention relate to oral vaccines introduced by consumption of atransgenic plant-derived antiviral vaccine. Such a plant derived vaccinemay take various forms including purified and partially purified plantderived viral antigen as well as whole plant, whole plant parts such asfruits, leaves, stems, tubers as well as crude extracts of the plant orplant parts. In general, the preferred state of the composition ofmatter which is used to induce an immune response (i.e., whole plant,plant part, crude plant exact, partially purified antigen or extensivelypurified antigen) will depend upon the ability of the immunogen toelicit a mucosal response, the dosage level of the plant derived antigenrequired to elicit a mucosal response, and the need to overcomeinterference of mucosal immunity by other substances in the chosencomposition of matter (i.e., sugars, pyrogens, toxins).

The present invention overcomes the deficiencies of the prior art byproducing oral vaccines in one or more tissues of a transgenic plant,thereby availing large human and animal populations of an inexpensivemeans of vaccine production and administration. In a preferredembodiment the edible fruit, juice, grain, leaves, tubers, stems, seeds,roots or other plant parts of the vaccine producing transgenic plant isingested by a human or an animal thus providing a very inexpensive meansof immunization against disease. In a preferred embodiment, such plantswill be plants routinely included in human and animal diets.Purification expense and adverse reactions inherent in existent vaccineproduction are thereby avoided. The invention also provides a novel andinexpensive source of antigen for more traditional vaccine deliverymodes. These and other aspects of the present invention will becomeapparent from the following description and drawings.

In one embodiment, the oral vaccine of the present invention is producedin edible transgenic plants and then administered through theconsumption of a part of those edible plants. A DNA sequence encodingthe expression of a surface antigen of a pathogen is isolated andligated into a plasmid vector containing selection markers. A promoterwhich regulates the production of the surface antigen in the transgenicplant is included in the same plasmid vector upstream from the surfaceantigen gene to ensure that the surface antigen is expressed in desiredtissues of the plant. Preferably, the foreign gene is expressed in aportion of the plant that is edible by humans or animals. For some uses,such as with human infants, it is preferred that the edible food be ajuice from the transgenic plant which can be taken orally.

In another embodiment, the vaccines (oral and otherwise) are provided byderiving recombinant viral antigens from the transgenic plants of theinvention in at least a semi-purified form prior to inclusion into avaccine. The present invention produces vaccines inexpensively. Further,vaccines from transgenic plants can not only be produced in theincreased quantity required for oral vaccines but can be administeredorally, thereby also reducing cost. The production of an oral vaccine inedible transgenic plants may avoid much of the time and expense requiredfor FDA approval and regulation relating to the purification of thevaccine.

A principal advantage of the present invention is the humanitarian goodwhich can be achieved through the production of inexpensive oralvaccines which can be used to vaccinate the populations of lesserdeveloped countries who otherwise could not afford expensive oralvaccines manufactured under present methods or vaccines which requireparenteral administration.

Thus, the invention provides for a recombinant mammalian viral proteinexpressed in a plant cell, which protein is known to elicit an antigenicresponse in a mammal in at least the native state of the virus.Preferably, the recombinant viral protein of the invention will also beone which is known to function as an antigen or immunogen (usedinterchangeably herein) as a recombinant protein when expressed instandard pharmaceutical expression systems such as yeasts or bacteria orwhere the viral protein is recovered from mammalian sera and shown to beantigenic. More preferably still, the antigenic/immunogenic protein ofthe invention will be a protein known to be antigenic/immunogenic whenthe protein as derived from the native virus, mammalian sera or fromstandard pharmaceutical expression systems, is used to induce the immuneresponse through an oral mode of introduction. In its most preferredembodiment, the recombinant mammalian viral protein, known to beantigenic in its native state, will be a protein which upon expressionin the plant cells of the invention, retains at least some portion ofthe antigenicity it possesses in the native state or as recombinantlyexpressed in standard pharmaceutical expression systems.

The immunogen of the invention is one derived from a mammalian virus andwhich is then expressed in a plant. In certain preferred embodiments,the mammalian virus from which the antigen is derived will be apathogenic virus of the mammal. Thus, it is anticipated that some of themost useful plant-expressed viral immunogens will be those derived froma pathogenic virus of a mammal such as a human.

The immunogens of the invention are preferably produced in plants whereat least a portion of the plant is edible. For the purposes of thisinvention, an edible plant or portion thereof is one which is not toxicwhen ingested by the mammal to be treated with the vaccine produced inthe plant. Thus, for instance, many of the common food plants will be ofparticular utility when used in the compositions and methods of theinvention. However, no nutritive value need be obtained when ingestingthe plants of the invention in order for such a plant to be includedwithin the types of the plants covered by the claimed invention.Moreover, in some cases, for instance in the domestic potato, a plantmay still be considered edible as used herein, although some tissues ofthe plant, but not the entire plant, may be toxic when ingested (i.e.,while potato tubers are not toxic and thus falling within thedefinitions of the claimed invention, the fruit of the potato is toxicwhen ingested). In such cases, such plants are still included within thedefinition of the claimed invention.

The immunogen of the invention, in a preferred embodiment, is a mucosalimmunogen. For the purposes of the invention, a mucosal immunogen is animmunogen which has the ability to specifically prime the mucosal immunesystem. In a more highly preferred embodiment, the mucosal immunogens ofthe invention are those mucosal immunogens which prime the mucosalimmune system and/or stimulate the humoral immune response in adose-dependent manner, without inducing systemic tolerance and withoutthe need for excessive doses of antigen. Systemic tolerance is definedherein as a phenomenon occurring with certain antigens which arerepeatedly fed to a mammal resulting in a specifically diminishedsubsequent anti-antigen response. Of course, while the immunogens of theinvention when used to induce a mucosal response may also induce asystemic tolerance, the same immunogen when introduced parenterally willtypically retain its immunogenicity without developing tolerance.

A mucosal response to the immunogens of the invention is understood toinclude any response generated when the immunogen interacts with amammalian mucosal membrane. Typically, such membranes will be contactedwith the immunogens of the invention through feeding of the immunogenorally to a subject mammal. Using this route of introduction of theimmunogen to the mucosal membranes provides access to the smallintestine M cells which overlie the Peyer's Patches and other lymphoidclusters of the gut-associated lymphoid tissue (GALT). However, anymucosal membrane accessible for contact with the immunogens of theinvention is specifically included within the definition of suchmembranes (e.g., mucosal membranes of the air passages accessible byinhaling, mucosal membranes of the terminal portions of the largeintestine accessible by suppository, etc.).

Thus, the immunogens of the invention may be used to induce both mucosalas well as humoral responses. Where the immunogens of the invention aresubjected to adequate levels of purification as further describedherein, these immunogens may be introduced parenterally such as bymuscular injection. Similarly, while preferred embodiments of theinvention include feeding of relatively unpurified immunogenpreparations (e.g., portions of edible plants, purees of such portionsof plants, etc.), the introduction of the immunogen to stimulate themucosal response may equally well occur through first subjecting theplant source of the immunogen to various purification proceduresdetailed herein or incorporated specifically by reference hereinfollowed by introduction of such a purified immunogen through any of themodes discussed above for accessing the mucosal membranes.

The recombinant immunogens of the invention may represent the entireamino acid sequence of the native immunogen of the virus from which itis derived. However, in certain embodiments of the invention, therecombinant immunogen may represent only a portion of the nativemolecule's sequence. In either case, the immunogen may be fused toanother peptide, polypeptide or protein to form a chimeric protein. Thefusion of the molecules is accomplished either post-translationallythrough covalent bonding of one to another (e.g., covalent bonding ofplant produced hepatitis B viral immunogen with whole hen egg lysozyme)or pre-translationally using recombinant DNA techniques (see e.g., supradiscussion of poli virus vaccines), both of which methods are known wellto those of skill in the art.

In certain embodiments, the immunogen of the invention will be animmunogen derived from a hepatitis virus. In particular embodiments, thehepatitis B virus surface antigen will be selected. Thus, in a highlypreferred embodiment, a viral mucosal immunogen derived from a hepatitisvirus is recombinantly expressed in a plant and is capable, in thenative state of the virus or as a recombinant protein expressed in anystandard pharmaceutical expression system, of eliciting an immuneresponse, particularly a mucosal immune response.

In other embodiments of the invention, a transgenic plant comprising aplant expressing a recombinant viral immunogen derived from a mammalianvirus is provided. For purposes of the invention, a transgenic plant isa plant expressing in at least some of the cells of the plant arecombinant viral immunogen. The transgenic plant of the invention, inpreferred embodiments, is an edible plant, where the immunogen is amucosal immunogen, or more preferably where a mucosal immunogen capableof binding a glycosylated molecule on the surface of a membrane of amucosal cell, and in some embodiments where the immunogen is a chimericprotein.

In other preferred embodiments, the transgenic plant of the inventionwill be a transgenic plant expressing a recombinant viral mucosalimmunogen of hepatitis virus, where the mucosal immunogen is capable ofeliciting an immune response, particularly a mucosal immune response, inthe native state of the virus or as derived from standard pharmaceuticalexpression systems.

Also claimed herein are compositions of matter known as vaccines, wheresuch vaccines are vaccines comprising a recombinant viral immunogenexpressed in a plant. For the purposes of the invention, a vaccine is acomposition of matter which, when contacted with a mammal, is capable ofeliciting an immune response. As described above, certain preferredvaccines of the invention will be those vaccines useful againstmammalian viruses as a mucosal immunogen, and more preferably asvaccines wherein the mucosal immunogen is capable of binding aglycosylated molecule on the surface of a membrane of a mucosal cell. Insome embodiments, the vaccine may comprise a chimeric protein immunogen.In other embodiments, the vaccine of the invention will comprise animmunogen derived from a hepatitis virus. In still other preferredembodiments, the vaccine of the invention will comprise a mucosalimmunogen of hepatitis virus expressed in a plant, where the mucosalimmunogen is capable of eliciting an immune response, particularly amucosal immune response, in the native state of the virus or as derivedfrom standard pharmaceutical expression systems.

A food composition is also provided by the invention which comprises atleast a portion of a transgenic plant capable of being ingested for itsnutritional value, said plant comprising a plant expressing arecombinant viral immunogen. For the purposes of the invention, a plantor portion thereof is considered to have nutritional value when itprovides a source of metabolizable energy, supplementary or necessaryvitamins or co-factors, roughage or otherwise beneficial effect uponingestion by the subject mammal. Thus, where the mammal to be treatedwith the food is an herbivore capable of bacterial-aided digestion ofcellulose, such a food might be represented by a transgenic monocotgrass. Similarly, although transgenic lettuce plants do notsubstantially contribute energy sources, building block molecules suchas proteins, carbohydrates or fats, nor other necessary or supplementalvitamins or cofactors, a lettuce plant transgenic for the viralimmunogen of a mammalian virus used as a food for that mammal would fallunder the definition of a food as used herein if the ingestion of thelettuce contributed roughage to the benefit of the mammal, even if themammal could not digest the cellulosic content of lettuce.

As described in the compositions of matter recited above, certainpreferred foods of the invention will include foods where the immunogenis a mucosal immunogen, or where mucosal immunogen is capable of bindinga glycosylated molecule on the surface of a membrane of a mucosal cell,or where the immunogen is a chimeric protein or where, the immunogen isan immunogen derived from a hepatitis virus. Thus, in a highly preferredembodiment, the food of the claimed invention will comprise at least aportion of a transgenic plant capable of being ingested for itsnutritional value, where the plant expresses a recombinant viral mucosalimmunogen of hepatitis virus, and where the mucosal immunogen is capableof binding a glycosylated molecule on a surface of a membrane of amucosal cell. In any case, the foods of the invention may be thoseportions of a plant including the fruit, leaves, stems, roots, or seedsof said plant.

Of particular importance to the compositions and methods of the claimedinvention are certain plasmid constructions useful in obtaining theplants, immunogens, vaccines, and foods of the invention. Thus, plasmidvectors for transforming a plant are claimed comprising a DNA sequenceencoding a mammalian viral immunogen and a plant-functional promoteroperably linked to the DNA sequence capable of directing the expressionof the immunogen in said plant. In certain embodiments, the plasmidvector further comprises a selectable or scorable marker gene tofacilitate the detection of the transformed cell or plant. In certainembodiments, plasmid vector of the invention will comprise the plantpromoter of cauliflower mosaic virus, CaMV35S. As with othercompositions of matter described above, certain preferred embodiments ofthe plasmid vector of the invention will be those where the planttransformed by the plasmid vector is edible, or where the immunogenencoded by the plasmid vector is a mucosal immunogen, or more preferablywhere the immunogen encoded by the plasmid vector is capable ofeliciting an immune response, particularly a mucosal immune response, inthe native state of the virus or as derived from standard pharmaceuticalexpression systems, or where the encoded immunogen is a chimericprotein, or where the encoded immunogen is an immunogen derived from ahepatitis virus. Thus, in a highly preferred embodiment, the plasmidvector of the invention useful for transforming a plant comprises a DNAsequence encoding a mucosal immunogen of hepatitis virus, where themucosal immunogen is capable of eliciting an immune response,particularly a mucosal immune response, in the native state of the virusor as derived from standard pharmaceutical expression systems and wherea plant-functional promoter is operably linked to the DNA sequencecapable of directing the expression of the immunogen in the plant. In avery similar embodiment, the invention provides for DNA fragments usefulfor microparticle bombardment transformation of a plant.

Methods for constructing tnansgenic plant cells are also provided by theinvention comprising the steps of constructing a plasmid vector or a DNAfragment by operably linking a DNA sequence encoding a viral immunogento a plant-functional promoter capable of directing the expression ofthe immunogen in the plant and then transforming a plant cell with theplasmid vector or DNA fragment. Where preferred, the method may beextended to produce transgenic plants from the transformed cells byincluding a step of regenerating a transgenic plant from the transgenicplant cell.

A method for producing a vaccine is also provided by the claimedinvention, comprising the steps of constructing a plasmid vector or aDNA fragment by operably linking a DNA sequence encoding a viralimmunogen to a plant-functional promoter capable of directing theexpression of the immunogen in the plant, transforming a plant cell withthe plasmid vector or DNA fragment, and then recovering the immunogenexpressed in the plant cell for use as a vaccine. Again, wherepreferred, the method provides for an additional step prior torecovering the immunogen for use as a vaccine, of regenerating atransgenic plant from the transgenic plant cell.

The recovery of the immunogen from the plant cell or whole plant maytake several embodiments. In one such embodiment, the method ofrecovering the immunogen of the invention is accomplished by obtainingan extract of the plant cell or whole plant or portions thereof. Inembodiments where whole plants are regenerated by the methods of theinvention, the recovery step may comprise merely harvesting at least aportion of the transgenic plant.

The methods of the invention provide for any of a number oftransformation protocols in order to transform the plant cells andplants of the invention. While certain preferred embodiments describedbelow utilize particular transformation protocols, it will be understoodby those of skill in the art that any transformation method may beutilized with in the definitions and scope of the invention. Suchmethods include microinjection, polyethylene glycol mediated uptake, andelectroporation. Thus, certain preferred methods will utilize anAgrobacterium transformation system, in particular, where theAgrobacterium system is an Agrobacterium tumefaciens-Ti plasmid system.In other preferred methods, the plant cell is transformed utilizing amicroparticle bombardment transformation system.

Plants of particular interest in the methods of the invention includetomato plants and tobacco plants as will be described in more detail inthe examples to follow. However, it will be understood by those of skillin the art of plant transformation that a wide variety of plant speciesare amenable to the methods of the invention. All such species areincluded within the definitions of the claimed invention including bothdicotyledon as well as monocotyledon plants.

As will be described in greater detail in the examples to follow, themethods of the invention by which plants are transformed may utilizeplasmid vectors which are binary vectors. In other embodiments, themethods of the invention may utilize plasmids which are integrativevectors. In a highly preferred embodiment, the methods of the inventionwill utilize the plasmid vector pB121.

Methods of administering any of the vaccines of the invention are alsoprovided. In certain general embodiments, such methods compriseadministering a therapeutic amount of the vaccine to a mammal. In morespecific embodiments, these methods entail introduction of the vaccineeither parenterally or non-parenterally into a mammalian subject. Wherea non-parenteral introduction mode is selected, certain preferredembodiments will comprise oral introduction of the vaccine into saidmammal. Whichever mode of introduction of the vaccine to the mammaliansubject is selected, it will be understood by those skilled in the artof vaccination that the selected mode must achieve vaccination at thelowest dose possible in a dose-dependent manner and by so doing elicitserun and/or secretory antibodies against the immunogen of the vaccinewith minimal induction of systemic tolerance. Where a mucosal route ofvaccination is selected, care should be taken to introduce the vaccineinto the gut lumen of the mammal at low dosages and in forms whichminimize the simultaneous introduction of interfering compounds such asgalactose and galactose-like saccharides.

In preferred embodiments, methods are provided by the invention ofadministering an edible portion of a transgenic plant, which transgenicplant expresses a recombinant viral immunogen, to a mammal as an oralvaccine against a virus from which said immunogen is derived. Thesemethods comprise harvesting at least an edible portion of the transgenicplant, and feeding the harvested plant or portion thereof to a mammal ina suitable amount to be therapeutically effective as an oral vaccine inthe mammal.

Similarly, the invention provides for methods of producing andadministering an oral vaccine, comprising the steps of constructing aplasmid vector or DNA fragment by operably linking a DNA sequenceencoding a viral immunogen to a plant-functional promoter capable ofdirecting the expression of the immunogen in a plant, transferring theplasmid vector into a plant cell, regenerating a transgenic plant fromthe cell, harvesting an edible portion of the regenerated transgenicplants, and feeding the edible portion of the plant to a mammal in asuitable amount to be therapeutically effective as an oral vaccine. Itis this embodiment that will be of particular utility in underdevelopedcountries committed to agricultural raw products as a main source ofmost necessities.

Other objects and advantages of the invention will appear from thefollowing description.

BRIEF DESCRIPTION OF THE DRAWINGS

For a detailed description of the preferred embodiment of the invention,reference will now be made to the accompanying drawings wherein:

FIG. 1 is a diagrammatic plasmid construct illustrating the constructionof the plasmid vector pHVA-1 containing the HBsAg gene for producing theHBsAg antigen in a plant; and

FIG. 2 is a map of the coding sequence for two structural genes andtheir regulatory elements in the plasmid pHVA-1; and

FIG. 3 is a diagrammatic plasmid construct illustrating the constructionof the plasmid vector pHB101 containing the HBsAg gene for producing theHBsAg antigen in a plant; and

FIG. 4 is a diagrammatic plasmid construct illustrating the constructionof the plasmid vector pHB102 containing the HBsAg gene for producing theHBsAg antigen in a plant; and

FIG. 5 is a map of the coding sequence for three structural genes andtheir regulatory elements in the plasmids pHB101 and pHB102; and

FIG. 6A indicates the HBsAg mRNA levels in transgenic tobacco plants;and

FIG. 6B indicates the HBsAg protein levels in transgenic tobacco plants;and

FIGS. 7A-7B is a micrograph of immunoaffinity purified rHBsAg with acorresponding histogram; and

FIG. 8 is a sucrose density gradient sedimentation of HBsAg fromtransgenic tobacco; and

FIG. 9 is a buoyant density gradient sedimentation of HBsAg fromtransgenic tobacco.

FIGS. 10A-10B is an RNA blot of transformed tomato leaf.

FIG. 11 is a tissue blot of tomato leaves.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention has several components which include: usingrecombinant DNA techniques to create a plasmid vector which contains aDNA segment encoding one or more antigenic proteins which conferimmunity in a human or an animal to a particular disease and for theexpression of antigenic protein(s) in desired tissues of a plant;selecting an appropriate host plant to receive the DNA segment encodingantigenic protein(s) and subsequently produce the antigenic protein(s);transferring the DNA segment encoding the antigenic protein(s) from theplasmid vector into the selected host plant; regenerating the transgenicplant thereby producing plants expressing the antigenic protein(s) whichfunctions as a vaccine(s); and administering an edible part of thetransgenic plant containing the antigenic protein(s) as an oral vaccineto either a human or an animal by the consumption of a tnansgenic plantpart. The present invention thereby provides for the production of atransgenic plant which when consumed as food, at least in part, by ahuman or an animal causes an immune response. This response ischaracterized by resistance to a particular disease or diseases. Theresponse is the result of the production in the transgenic plant ofantigenic protein(s). The production of the antigenic protein(s) is theresult of stable genetic integration into the transgenic plant of DNAregions designed to cause regulated expression of antigenic protein(s)in the transgenic plants.

Vaccine(s) and Their Administration

The present invention may be used to produce any type vaccine effectivein immunizing humans and animals against diseases. Viruses, bacteria,fungi, and parasites that cause disease in humans and animals cancontain antigenic protein(s) which can confer immunity in a human or ananimal to the causative pathogen. A DNA sequence encoding any of theseviral, bacterial, fungal or parasitic antigenic proteins may be used inthe present invention.

Mutant and variant forms of the DNA sequences encoding a antigenicprotein which confers immunity to a particular virus, bacteria, fungusor parasite in an animal (including humans) may also be utilized in thisinvention. For example, expression vectors may contain DNA codingsequences which are altered so as to change one or more amino acidresidues in the antigenic protein expressed in the plant, therebyaltering the antigenicity of the expressed protein. Expression vectorscontaining a DNA sequence encoding only a portion of an antigenicprotein as either a smaller peptide or as a component of a new chimericfusion protein are also included in this invention.

The present invention is advantageously used to produce viral vaccinesfor humans and animals. The following table sets forth a list ofvaccines now used for the prevention of viral diseases in humans.

    ______________________________________                           Condition  Route of    Disease Source of Vaccine                           of Virus   Administration    ______________________________________    Poliomyelitis            Tissue culture Live attenuated                                      Oral            (human diploid cell                           Killed     Subcutaneous            line, monkey kidney)    Measles Tissue culture Live attenuated                                      Subcutaneous            (chick embryo)    Mumps   Tissue culture Live attenuated                                      Subcutaneous            (chick embryo)    Rubella Tissue culture Live attenuated                                      Subcutaneous            (duck embryo, rabbit,            or human diploid)    Smallpox            Lymph from calf or                           Live vaccinia                                      Intradermal            sheep    Yellow  Tissue cultures and eggs                           Live attenuated                                      Subcutaneous    Fever    Viral   Purified HBsAg from                           Live attenuated                                      Subcutaneous    hepatitis B            "health" carriers            Recombinant HBsAg                           Subunit    Subcutaneous            from yeast    Influenza            Highly purified                           Killed     Subcutaneous            or subviral            forms (chick embryo)    Rabies  Human diploid  Killed     Subcutaneous            cell cultures    Adenoviral            Human diploid  Live attenuated                                      Oral    infections            cell cultures    Japanese B            Tissue culture Killed     Subcutaneous    encephalitis            (hamster kidney)    Varicella            Human diploid  Live attenuated                                      Subcutaneous            cell cultures    ______________________________________

The present invention is also advantageously used to produce vaccinesfor animals. Vaccines are available to immunize pets and productionanimals. Diseases such as: canine distemper, rabies, canine hepatitis,parvovirus, and feline leukemia may be controlled with properimmunization of pets. Viral vaccines for diseases such as: Newcastle,Rinderpest, hog cholera, blue tongue and foot-mouth can control diseaseoutbreaks in production animal populations, thereby avoiding largeeconomic losses from disease deaths. Prevention of bacterial diseases inproduction animals such as: brucellosis, fowl cholera, anthrax and blackleg through the use of vaccines has existed for many years. Today newrecombinant DNA vaccines, e.g. rabies and foot and mouth, have beensuccessfully produced in bacteria and yeast cells and can facilitate theproduction of a purified vaccine containing only the immunizing antigen.Veterinary vaccines utilizng cloned antigens for protozoans andhelminths promise relief from parasitic infections which cripple andkill.

The oral vaccine produced by the present invention is administered bythe consumption of the foodstuff which has been produced from thetransgenic plant producing the antigenic protein as the vaccine. Theedible part of the plant is used as a dietary component while thevaccine is administered in the process.

The present invention allows for the production of not only a singlevaccine in an edible plant but for a plurality of vaccines into onefoodstuff. DNA sequences of multiple antigenic proteins can be includedin the expression vector used for plant transformation, thereby causingthe expression of multiple antigenic amino acid sequences in onetransgenic plant. Alternatively, a plant may be sequentially orsimultaneously transformed with a series of expression vectors, each ofwhich contains DNA segments encoding one or more antigenic proteins. Forexample, there are five or six different types of influenza, eachrequiring a different vaccine. A transgenic plant expressing multipleantigenic protein sequences can simultaneously elicit an immune responseto more than one of these strains, thereby giving disease immunity eventhough the most prevalent strain is not known in advance.

Vaccines produced in accordance with the present invention may also beincorporated into the feed of animals. This represents an importantmeans to produce lower cost disease prevention for pets, productionanimals, and wild species.

While the vaccines of the present invention be preferably utilizeddirectly as oral vaccines of the transgenic plant material, immunogeniccompositions derived from the transgenic plant materials suitable foruse as more traditional immune vaccines may be readily prepared from thetransgenic plant materials described herein. Preferably, such immunecompositions will comprise a material purified from the transgenicplant. Purification of the antigen may take many forms known well tothose of skill in the art, in particular such purifications will likelytrack closely the purification techniques used successfully in obtainingviral antigen particles from recombinant yeasts (i.e., those containingHBsAg). In one embodiment, detailed in the examples to follow, HBsAgviral protein-containing particles, similar in many respects to thoseobtained from recombinant yeasts, were purified from transformed tobaccoplants using a particular purification procedure. Whatever initialpurification scheme is utilized, the purified material will also beextensively dialyzed to remove undesired small molecular weightmolecules (i.e., sugars, pyrogens) and/or lyophilization of the thuspurified material for more ready formulation into a desired vehicle.

The preparation of vaccines is generally well understood in the art(e.g., those derived from fermentative yeast cells known well in the artof vaccine manufacture cite to Valenzuela et al Nature 298, 347-350(1982), as exemplified by U.S. Pat. Nos.4,608,251; 4,601,903; 4,599,231;4,599,230; 4,596,792; and 4,578,770, all incorporated herein byreference. Typically, such vaccines are prepared as injectables, eitheras liquid solutions or suspensions. Solid forms suitable for solutionin, or suspension in, liquid prior to injection may also be prepared.

The preparation may also be emulsified. The active immunogenicingredient is often mixed with excipients which are pharmaceuticallyacceptable and compatible with the active ingredient. Suitableexcipients are, for example, water, saline, dextrose, glycerol, ethanol,or the like and combinations thereof. In addition, if desired, thevaccine may contain minor amounts of auxiliary substances such aswetting or emulsifying agents, pH buffering agents, or adjuvants whichenhance the effectiveness of the vaccines.

The vaccines are conventionally administered parenterally, by injection,for example, either subcutaneously or intramuscularly. Additionalformulations which are suitable for other modes of administrationinclude suppositories and, in some cases, oral formulations or aerosols.For suppositories, traditional binders and carriers may include, forexample, polyalkalene glycols or triglycerides: such suppositories maybe formed from mixtures containing the active ingredient in the range of0.5% to 10%, preferably 1-2%. Oral formulations other than edible plantportions described in detail herein include such normally employedexcipients as, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate and the like. These compositions take the form of solutions,suspensions, tablets, pills, capsules, sustained release formulations orpowders and contain 10-95% of active ingredient, preferably 25-70%.

In many instances, it will be desirable to have multiple administrationsof the vaccine, usually not exceeding six vaccinations, more usually notexceeding four vaccinations and preferably one or more, usually at leastabout three vaccinations. The vaccinations will normally be at from twoto twelve week intervals, more usually from three to five weekintervals. Periodic boosters at intervals of 1-5 years, usually threeyears, will be desirable to maintain protective levels of theantibodies.

The course of the immunization may be followed by assays for antibodiesfor the supernatant antigens. The assays may be performed by labelingwith conventional labels, such as radionuclides, enzymes, fluorescers,and the like. These techniques are well known and may be found in a widevariety of patents, such as U.S. Pat. Nos. 3,791,932; 4,174,384 and3,949,064, as illustrative of these types of assays.

Host Plant Selection

A variety of plant species have been genetically transformed withforeign DNA, using several different gene insertivetechniques.¹⁰,22-27,29-32 Since important progress is being made toclone DNA coding regions for vaccine antigens for parasitic tropicaldiseases and veterinary parasitic diseases¹¹⁻²¹ the present invention,will have important means of low cost production of vaccines in a formeasily used for animal treatment.

Since many edible plants used by humans for food or as components ofanimal feed are dicotyledenous plants, it is preferred to employdicotyledons in the present invention, although monocotyledontransformation is also applicable especially in the production ofcertain grains useful for animal feed.

The host plant selected for genetic transformation preferably has edibletissue in which the antigenic protein, a proteinaceous substance, can beexpressed. Thus, the antigenic protein is expressed in a part of theplant, such as the fruit, leaves, stems, seeds, or roots, which may beconsumed by a human or an animal for which the vaccine is intended.Although not preferred, a vaccine may be produced in a nonedible plantand administered by one of various other known methods of administeringvaccines.

Various other considerations are made in selecting the host plant. It issometimes preferred that the edible tissue of the host plant not requireheating prior to consumption since the heating may reduce theeffectiveness of the vaccine for animal or human use. Also, sincecertain vaccines are most effective when administered in the human oranimal infancy period, it is sometimes preferred that the host plantexpress the antigenic protein which will function as a vaccine in theform of a drinkable liquid.

Plants which are suitable for the practice of the present inventioninclude any dicotyledon and monocotyledon which is edible in part or inwhole by a human or an animal such as, but not limited to, carrot,potato, apple, soybean, rice, corn, berries such as strawberries andraspberries, banana and other such edible varieties. It is particularlyadvantageous in certain disease prevention for human infants to producea vaccine in a juice for ease of administration to humans such as tomatojuice, soy bean milk carrot juice, or a juice made from a variety ofberry types. Other foodstuffs for easy consumption might include driedfruit.

Methods of Gene Transfer into Plants

There are various methods of introducing foreign genes into bothmonocotyledenous and dicotyledenous plants.³³,34 The principle methodsof causing stable integration of exogenous DNA into plant genomic DNAinclude the following approaches: 1) Agrobacterium--mediated genetransfer,³⁵,36,37,53 2) direct DNA uptake,³⁸ including methods fordirect uptake of DNA into protoplasts,⁸ DNA uptake induced by briefelectric shock of plant cells,⁴¹,42 DNA injection into plant cells ortissues by particle bombardment,³⁹,44-45 by the use of micropipettesystems,⁴³,47,48 or by the direct incubation of DNA with germinatingpollen;⁴⁰,49 or 3) the use of plant virus as gene vectors.³³,51

The Agrobacterium system includes the use of plasmid vectors thatcontain defined DNA segments that integrate into the plant genomic DNA.Methods of inoculation of the plant tissue vary depending upon the plantspecies and the Agrobacterium delivery system. A widely used approach isthe leaf disc procedure which can be performed with any tissue explantthat provides a good source for initiation of whole plantdifferentiation.⁶ The Agrobacterium system is especially viable in thecreation of transgenic dicotyledenous plants.

As listed above there are various methods of direct DNA transfer intoplant cells. In electroporation, the protoplasts are briefly exposed toa strong electric field. In microinjection, the DNA is mechanicallyinjected directly into the cells using very small micropipettes. Inmicroparticle bombardment, the DNA is adsorbed on microprojectiles suchas magnesium sulfate crystals or tungsten particles, and themicroprojectiles are physically accelerated into cells or plant tissues.

The last principle method of vector transfer is the transmission ofgenetic material using modified plant viruses. DNA of interest isintegrated into DNA viruses, and these viruses are used to infect plantsat wound sites.

In the preferred embodiment of the present invention, theAgrobacterium-Ti plasmid system is utilized.⁵³ The tumor-inducing (Ti)plasmids of A. tumefaciens contain a segment of plasmid DNA calledtransforming DNA (T-DNA) which is transferred to plant cells where itintegrates into the plant host genome. The construction of thetransformation vector system has two elements. First, a plasmid vectoris constructed which replicates in Escherichia coli (E. coli). Thisplasmid contains the DNA encoding the protein of interest (an antigenicprotein in this invention); this DNA is flanked by T-DNA bordersequences that define the points at which the DNA integrates into theplant genome. Usually a gene encoding a selectable marker (such as agene encoding resistance to an antibiotic such as Kanamycin) is alsoinserted between the left border (LB) and right border (RB) sequences;the expression of this gene in transformed plant cells gives a positiveselection method to identify those plants or plant cells which have anintegrated T-DNA region.⁵²,53 The second element of the process is totransfer the plasmid from E. coli to Agrobacterium. This can beaccomplished via a conjugation mating system, or by direct uptake ofplasmid DNA by Agrobacterium. For subsequent transfer of the T-DNA toplants, the Agrobacterium strain utilize must contain a set of induciblevirulence (vir) genes which are essential for T-DNA transfer to plantcells.⁵³,54

Those skilled in the art should recognize that there are multiplechoices of Agrobacterium strains and plasmid construction strategiesthat can be used to optimize genetic transformation of plants. They willalso recognize that A. tumefaciens may not be the only Agrobacteriumstrain used. Other Agrobacterium strains such as A. rhizogenes might bemore suitable in some applications.

Methods of inoculation of the plant tissue vary depending upon the plantspecies and the Agrobacterium delivery system. A very convenientapproach is the leaf disc procedure which can be performed with anytissue explant that provides a good source for initiation of whole plantdifferentiation. The addition of nurse tissue may be desirable undercertain conditions. Other procedures such as the in vitro transformationof regenerating protoplasts with A. tumefaciens may be followed toobtain transformed plant cells as well.³³,53

This invention is not limited to the Agrobacterium-Ti plasmid system butshould include any direct physical method of introducing foreign DNAinto the plant cells, transmission of genetic material by modified plantviruses, and any other method which would accomplish foreign DNAtransfer into the desired plant cells.

Promoters

Once the host plant has been selected and the method of gene transferinto the plant determined, a constitutive, a developmentally regulated,or a tissue specific promoter for the host plant is selected so that theforeign protein is expressed in the desired part(s) of the plant.

Promoters which are known or found to cause transcription of a foreigngene in plant cells can be used in the present invention. Such promotersmay be obtained from plants or viruses and include, but are notnecessarily limited to: the 35S promoter of cauliflower mosaic virus(CaMV) (as used herein, the phrase "CaMV 35S" promoter includesvariations of CaMV 35S promoter, e.g. promoters derived by means ofligations with operator regions, random or controlled mutagenesis,etc.); promoters of seed storage protein genes such as Zma10Kz or Zmag12(maize zein and glutelin genes, respectively), light-inducible genessuch as ribulose bisphosphate carboxylase small subunit (rbcS), stressinduced genes such as alcohol dehydrogenase (Adhl), or "housekeepinggenes" that express in all cells (such as Zmaact, a maize actingene).⁴,55 This invention can utilize promoters for genes which areknown to give high expression in edible plant parts, such as the patatingene promoter from potato.⁵⁶

The plasmid constructed for plant transformation also usually contains aselectable or scorable marker gene. Numerous genes for this purpose havebeen identified.⁵⁴,57

The following are examples of the production of a vaccine for hepatitisB in a host transgenic tomato and tobacco plant and are presented todescribe a preferred embodiment and the utility of the present inventionbut should not be construed as limiting the claims thereof.

The DNA coding sequence for the hepatitis B surface antigen was selectedfor expression in a transgenic plant as Hepatitis B virus is one of themost widespread viral infections of humans which causes acute andchronic hepatitis and heptocellular carcinoma.⁷¹ Tomato and tobaccoplants were selected as the host plants to produce the hepatitis Brecombinant surface antigen as examples of antigenic protein productionin different plant parts. Expression of HbsAg in tobacco and tomatoplants was accomplished by the method of Mason, H. S. Lam, and Arntzen,C. J., Proceedings of the National Academy of Sciences, U.S.A. Vol. 89,11745-11749(1992), herein incorporated by reference.

EXAMPLE I

A. Construction of Hepatitis B Surface Antigen Expression Vector pHVA-1

Referring initially to the diagrammatic plasmid construct illustrated inFIG. 1, the DNA sequence encoding for HBsAg contained within restrictionendonuclease sites Pst I-Hind III on plasmid pWR/HBs-3 was excised andsubsequently ligated into the unique Bam H-Sst I site of the excisedbeta-glucuronidase (GUS) gene on plasmid pB121 to construct the binaryvector plasmid pHVA-1.

Plasmid pB 121, obtained from Clonetech Laboratories, Inc., Palo Alto,Calif., has cleavage sites for the restriction endonucleases Bam HI andSst I located between the CaMV 35S promoter and the GUS structural geneinitiation sequence and between the GUS gene termination sequence andthe NOS polyadenylation signals, respectively. Plasmid pB121 wasselected since the GUS structural gene can be excised from the plasmidusing Bam HI and Sst I, another structural gene encoding an antigenicprotein can be inserted, and the new gene will be functionally active inplant gene expression. Plasmid pB121 also contains a NPT II geneencoding neomycin phosphotransferase II; this is an enzyme that confersKanamycin resistance when expressed in transformed plant cells, therebyallowing the selection of cells and tissues with integrated T-DNA. TheNPT II gene is flanked by promoter and polyadenylation sequences from aNopaline synthase (NOS) gene.

The HBsAg DNA coding sequence⁶⁴,65 was isolated from the plasmidpWR/BBs-3 (constructed at the Institute of Cell Biology in China) as aPst I--Hind III fragment. This fragment was digested with Klenow enzymeto create blunt ends; the resultant fragment was ligated at the 5' endwith Bam H1 linkers and at the 3' end with Sst 1 linkers, and theninserted into the pB 121 plasmid at the site where the GUS codingsequence had been excised, thereby creating plasmid pHVA-1 as shown inFIG. 1.

The plasmid vector pHVA-1 then contains 1) a neomycin phosphotransferaseII (NPT II) gene which provides the selectable marker for kanamycinresistance; 2) a HBsAg gene regulated by a cauliflower mosaic virus(CaMV 35S) promoter sequence; and 3) right and left T-DNA bordersequences which effectively cause the DNA sequences for the NOS andHBsAg genes to be transferred to plant cells and integrated into theplant genome. The diagrammatic structure of pHVA-1 is shown in FIG. 2.

B. Transfer of Binary Vector, pHVA-1, to A. tumefaciens

Plasmid pHVA-1, containing the HBsAg gene, was transferred to A.tumefaciens strain LBA4404 obtained from Clontech Laboratories, Inc.This strain is widely used since it is "disarmed"; that is, it hasintact vir genes, but the T-DNA region has been removed by in vivodeletion techniques. The vir genes work in trans to mediate T-DNAtransfer to plants from the plasmid pHVA-1.

A. tumefaciens was cultured in AB medium⁵⁸ containing two-tenthsmilligrams per milliliter (0.2 mg/ml) streptomycin until the opticaldensity (O.D.) at six hundred nanometers (600 nm) of the culture reachesabout five tenths (0.5). The cells are then centrifuged at 2000 timesgravity (2000×G) to obtain a bacterial cell pellet. The Agrobacteriumpellet was resuspended in one milliliter of ice cold twenty millimolarcalcium chloride (20 mM CaCI₂). Five tenths microgram (0.5 □g) ofplasmid pHVA-1 DNA was added to two tenths milliliters (0.2 ml) of thecalcium chloride suspension of A. tumefaciens cells in a one and fivetenths milliliter (1.5 ml) microcentrifuge tube and incubated on ice forsixty minutes. The plasmid pHVA-1 DNA and A. tumefaciens cells mixturewas frozen in liquid nitrogen for one minute, thawed in a twenty-fivedegree Celsius (25□C) water bath, and then mixed with five volumes orone milliliter (1 ml) of rich MGL medium.⁵⁸ The plasmid pHVA-1 and A.tumefaciens mixture was then incubated at twenty-five degrees Celsius(25□C) for four hours with gentle shaking. The mixture was plated on LB,luria broth,⁵⁸ agar medium containing fifty micrograms per milliliter(50 □g/ml) kanamycin. Optimum drug concentration may differ dependingupon the Agrobacterium strain in other experiments. The plates wereincubated for three days at twenty-five degrees Celsius (25□C) beforeselection of resultant colonies which contained the transformedAgrobacterium harboring the pHAV-1 plasmids.

The presence of pHVA-1 DNA in the transformed Aerobacterium culture wasverified by restriction mapping of the plasmid DNA purified by alkalinelysis of the bacterial cells.⁵⁹

Plant Transformation by A. tumefaciens Containing the UBsAg Gene as Partof the Ti Vector System

The technique for in vitro transformation of plants by theAgrobacterium-Ti plasmid system is based on cocultivation of planttissues or cells and the transformed Agrobacterium for about two dayswith subsequent transfer of plant materials to an appropriate selectivemedium. The material can be either protoplast, callus or organ tissue,depending upon the plant species. Organ cocultivation with leaf piecesis a convenient method.

Leaf disc transformation was performed in accordance with the procedureof Horsch et al⁶. Tomato and tobacco seedlings were grown in flats undermoderate light and temperature and low humidity to produce uniform,healthy plants of ten to forty centimeters in height. New flats werestarted weekly and older plants were discarded. The healthy, unblemishedleaves from the young plants were harvested and sterilized in bleachsolution containing ten per cent (10%) household bleach (diluted one toten from the bottle) and one tenth per cent (0.1%) Tween 20 or othersurfactant for fifteen to twenty minutes with gentle agitation. Theleaves were then rinsed three times with sterile water. The leaf discswere then punched with a sterile paper punch or cork borer, or cut intosmall strips or squares to produce a wounded edge.

Leaf discs were precultured for one to two days upside down on MS 104⁶medium to allow initial growth and to eliminate those discs that weredamaged during sterilization or handling. Only the leaf discs whichshowed viability as evidenced by swelling were used for subsequentinoculation. The A. tumefaciens containing pHVA-1 which had been grownin AB medium were diluted one to twenty with MSO⁶ for tomato inoculationand one to ten for tobacco discs. Leaf discs were inoculated byimmersion in the diluted transformed A. tumefaciens culture andcocultured on regeneration medium MS 104⁶ medium for three days. Leafdiscs were then washed with sterile water to remove the free A.tumefaciens cells and placed on fresh MS selection medium whichcontained three hundred micrograms per milliliter (300 □g/ml) ofkanamycin to select for transformed plants cells and five hundredmicrograms per milliliter (500 □g/nml) carbenicillin to kill anyremaining A. tumefaciens. The leaf discs were then transferred to freshMS selection medium at two week intervals. As shoots formed at the edgeof the leaf discs and grew large enough for manual manipulation, theywere excised (usually at three to six weeks after cocultivation withtransformed A. tumefaciens) and transferred to a root-inducing medium,e.g. MS rooting medium.⁶ As roots appeared the plantlets were eitherallowed to continue to grow under sterile tissue culture conditions ortransferred to soil and allowed to grow in a controlled environmentchamber.

D. Selection of Genetically-Engineered Plants Which Express HBsAg

Approximately three months (nine months for tomato fruit assays) afterthe initial cocultivation of the putative BBsAg expressing tomato plants(HB-plants) with A. tumefaciens, they were tested for the presence ofHBsAg.

1. Biochemical and Immunochemical Assays

Root, stem, leaf and fruit samples of the plants were excised. Eachtissue was homogenized in a buffered solution, e.g. one hundredmillimolar sodium phosphate (100 mM), pH 7.4 containing one millimolarethylenediamine tetraacetate (1.0 mM EDTA) and five-tenths millimolarphenylmethylsulfonyl fluoride (0.5 mM PMSF) as a proteinase inhibitor.The homogenate was centrifuged at five thousand times gravity (5000×G)for ten minutes. A small aliquot of each supernatant was then reservedfor protein determination by the Lowry method. The remaining supernatantwas used for the determination of the level of HBsAg expression usingtwo standard assays: (a) a HBsAg radioimmunoassay, the reagents forwhich were purchased from Abbott Laboratories and (b) immunoblottingusing a previously described method of Peng and Lam⁶¹ with a monoclonal10 antibody against anti-HBsAg purchased from Zymed Laboratories.Depending upon the level of HBsAg expression in each tissue, thesupernatant may have been partially purified using a previouslydescribed affinity chromatographic method of Pershing et al⁶² usingmonoclonal antibody against HBsAg bound to commercially availableAffi-Gel 10 gel from Bio-Rad Laboratories, Richmond, Calif. The purifiedsupernatant was then concentrated by lyophilization or ultrafiltrationprior to radioimmunoassay and immunoblotting.

2. Detection of the HBsAg Gene Construct

The stable integration of the HBsAg construct (expression vector) forplant cell transfection was tested by hybridization assays of genomicDNA digested with Eco R1, and with a combined mixture of Bam H1 and Sst1 in each plant tissue for both control and HBsAg-transfected plantswith a HBsAg coding sequence probe using standard southern blots⁶⁰. Inaddition, seeds were collected from self-fertlized plants, and progenywere analyzed by standard Southern analysis.

E. Regeneration of HBsAg Transgenic Tomato Plants

Once the transgenic plant has been perfected, the transgenic plant isregenerated by growing multiples of the transgenic plant to produce theoral vaccine. Of course, the most common method of plant propagation isby seed. Regeneration by seed propagation, however, has the deficiencythat there is a lack of uniformity in the crop. Seeds are produced byplants according to the genetic variances governed by Mendelian rules.Basically, each seed is genetically different and each will grow withits own specific traits. Therefore, it is preferred that the transgenicplant be produced by homozygous selection such that the regeneratedplant has the identical traits and characteristics of the parenttransgenic plant, e.g. a reproduction of the vaccine.

F. Administration of HBsAg Vaccine to Humans Through Consumption ofTomato Juice Produced from HBsAg Transgenic Tomatoes

Once the vaccine is produced through the mass regeneration of thetransgenic plant, the crop is harvested and utilized directly as food orprocessed into a consumable food. Although the food may be processed asa solid or liquid, in some cases it is preferred that it be in liquidform for ease of consumption. The transgenic tomatoes could behomogenized to produce tomato juice which could be bottled for drinking.HBsAg vaccine administration is accomplished by a human drinking thetomato juice or consuming the fruit in a quantity and time scale (onceor multiple doses over a period of time) to confer immunity to hepatitisB virus infection.

EXAMPLE II

A.1 Construction of Hepatitis B Surface Antigen Expression Vector pHB101

Referring to the plasmid construct illustrated in FIG. 3, the DNAsequence encoding for HBsAg contained within restriction endonucleasesites Pst I-Hind IH on plasmid pMT-SA (provided by Li-he Guo, ChineseAcademy of Sciences) was excised and subsequently ligated into theunique Bam HI-Sac I site of the excised beta-Glucuronidase (GUS) gene onplasmid pBI121 to construct the binary plasmid pB 101.

Plasmid pBI121, obtained from Clonetech Laboratories, Inc., Palo Alto,Calif., has cleavage sites for the restriction endonucleases Bam HI andSac I located between the CaMV 35S promoter and the GUS structural geneinitiation sequence and between the GUS gene termination sequence andthe NOS polyadenylation signals, respectively. Plasmid pBI121 wasselected since the GUS structural gene can be excised from the plasmidusing Bam HI and Sac T. another structural gene encoding an antigenicprotein can be inserted, and the new gene will be functionally active inplant gene expression. Plasmid pBI121 also contains a NPT II geneencoding neomycin phosphotransferase R and conferring kanamycinresistance. The NPT II gene is flanked by promoter and polyadenylationsequences from a Nopaline synthase (NOS) gene.

The HBsAg DNA coding sequence⁶⁴,65 (the S gene) was excised from plasmidpMT-SA (constructed at Chinese Academy of Sciences) as a Pst I-Hind mHfragment and isolated by electrophoresis in a one percent (1%) agarosegel. The Pst-Hind mH fragment was visualized in the agarose gel bystaining with ethidium bromide, illuminated with ultraviolet light (UV)and purified with a Prep-aGene kit (BioRad Laboratories, Richmond,Calif.). The HBsAg coding region on the Pst I-Hind III fragment was thenligated into the Pst I-Hind mH digested plasmid pBluescript KS(Stratagene, La Jolla, Calif.) to form the plasmid pKS-FBS. The HBsAggene in plasmid pKS-HBS was then opened 116 base pairs (bp) 3' to thetermination codon with BstB I and the resulting ends were blunted byfilling with Klenow enzyme and dCTP/dGTP. The entire coding region (820bp) was then excised with Bam HI, which is site derived from the plasmidvector pBluescript. This results in the addition of Bam HI and Sma Isites 5' to the original HBsAg coding sequence from plasmid pMT-SA.

Plasmid pBI121⁶⁶, obtained from Clonetech, Laboratories, Inc., PaloAlto, Calif., was digested with Sac I and the ends blunted with mungbean nuclease. The GUS coding region was then released from pBI121 bytreatment with Bam HI and the 11 kilobase pair (kbp) GUS-less pBI121plasmid vector isolated. Subsequently, the HBsAg coding fragment excisedfrom pKS-HB was ligated into the GUS-less plasmid pBI121 to yieldplasmid pHB101 (FIG. 3). Transcription of the HBsAg gene in thisconstruct is driven by the cauliflower mosaic virus 35S (CaMV 35S)promoter derived from pBI121, and the polyadenylation signal is providedby the nopaline synthase terminator.

The plasmid vector pHB101 then contains 1) a neomycin phosphotransferaseII (NPTII) gene which provides the selectable marker for kanamycinresistance; 2) a HBsAg gene regulated by a cauliflower mosaic virus(CaMV 35S) promoter sequence; and 3) right and left T-DNA bordersequences which effectively cause the DNA sequences for the NOS andHBsAg genes to be transferred to plant cells and integrated into theplant genome. The diagrammatic structure of pHB101 is shown in FIG. 5.

A.2 Construction of Hepatitis B Surface Antigen Expression Vector pHB102

Plasmid pHB102, an improved expression vector, was constructed fromplasmid pHB101 by removal of the CaMV 35S promoter and insertion of amodified 35S promoter linked to a translational enhancer element. TheCaMV 35S promoter in the plasmid pRTL2-GUS⁶⁷ contains a duplication ofthe upstream regulatory sequences between nucleotides -340 and -90relative to the transcription initiation site. Fused to the 3' end ofthe promoter is the tobacco etch virus 5' nontranslated leader sequence(TL), which acts as a translational enhancer in tobacco cells.

As seen in FIG. 4, the promoter (with dual enhancer) was obtained fromplasmid pRTL2GUS. pRTL2GUS was digested with Nco I and the ends wereblunted with mung bean nuclease. The CaMV 35S with duplicated enhancerlinked to tobacco etch virus (IEV) 5' nontranslated leader sequence (thepromoter-leader fragment) was then released by digestion with Hind Im,and purified by agarose gel electrophoresis. Plasmid pHB101 was digestedwith Hind Im and Sma I to release the CaMV 35S promoter fragment and thepromoterless plasmid vector was purified by agarose gel electrophoresis.This yielded a blunt end just 5' to the HbsAg coding sequence for fusionwith the blunted Nco I site at the 3' end of the purifiedpromoter-leader fragment from pRTL2GUS. Then the promoter-leaderfragment from pRTL2GUS was ligated into the Hind III-Sma I site onpromoter-less plasmid pHB101 to yield plasmid pHB102.

The HBsAg coding region of plasmid pHB102 lies upstream of the nopalinesynthase (NOS) terminator. The plasmid contains the left and rightborders of the T-DNA that is integrated into the plant genomic DNA viaAgrobacterium tumefaciens mediated transformation, as well as theneomycin phosphotransferase (NPT II) gene which allows selection withkanamycin. Expression of the HbsAg gene is driven by the CaMV 35S withdual transcriptional enhancer linked to the TEV 5' nontranslated leader.The TEV leader acts as a translational enhancer to increase the amountof protein made using a given amount of template MRNA.⁶⁷

B. Transfer of Binary Vectors, pHB101 and pHB102, to A. tumefaciens

Plasmid pHB101, containing the HbsAg gene and the CaMV 35S promoter, andplasmid pHB102, containing the HBsAg gene and CaMV 35S promoter withdual transcription enhancer linked to the TEV 5' nontranslated leaderwere then separately transferred to Agrobacterium tumefaciens. PlasmidpHB101 or pHB102, each containing the HBsAg gene, was transferred to theA. tumefaciens strain LBA4404 obtained from Clonetech Laboratories, Inc.as in Example I.

A. tumefaciens was cultured in 50 milliliters (50 ml) of YEP (yeastextract-peptone broth)⁵⁸ containing two-tenths milligrnms per milliliter(0.2 mg/mi) streptomycin until the optical density (O.D.) at 600nanometers (nm) of the culture reaches about five tenths (0.5). Thecells were then centrifuged at 2000 times gravity (2000×G) to obtain abacterial cell pellet. The Agrobacterium pellet was resuspended in tenmilliliters of ice cold one hundred fifty millimolar sodium chloride(150 mM NaCl₂). The cells were then centrifuged again at 2000×G and theresulting Agrobacterium pellet was resuspended in one milliliter (1 ml)of ice cold twenty millimolar calcium chloride (20 mM CaCl₂).Five-tenths microgram (0.5 □g) of plasmid pHB101 or plasmid pHB102 wasadded to two tenths milliliters (0.2 ml) of the calcium chloridesuspension of A. tumefaciens cells in a one and five tenths milliliter(1.5 ml) microcentrifuge tube and incubated on ice for sixty minutes.The plasmid pHB110 or pHB102 DNA and A. tumefaciens cells mixture wasfrozen in liquid nitrogen for one minute, thawed in a twenty-eightdegree Celsius (28□C) water bath, and then mixed with five volumes or 1milliliter (1 ml) of YEP (yeast extract-peptone broth). The plasmidpHB101 or pHB102 and A. tumefaciens mixture was then incubated attwenty-eight degrees Celsius (28□C) for four hours with gentle shaking.The mixture was plated on YEP (yeast extract-peptone broth) agar mediumcontaining fifty micrograms per milliliter (50 □g/ml) kanamycin. Optimumdrug concentration may differ depending upon the Agrobacterium strain inother experiments. The plates were incubated for three days attwenty-eight degrees Celsius (28□C) before selection of resultantcolonies which contained the transformed Agrobacterium harboring thepHB101 or the pHB102 plasmids. These colonies were then transferred tofive milliliters (5 ml) of YEP (yeast extract-peptone broth) containingfifty micrograms per milliliter (50 □g/ml) of kanamycin for three daysat twenty-eight degrees Celsius (28□C).

The presence of pHB101 or pHB102 DNA in the transformed Agrobacteriumculture was verified by restriction mapping of the plasmid DNA purifiedby alkaline lysis of the bacterial cells.⁵⁹

C. Plant Transformation by A. tumefaciens containing the HBsAg Gene asPart of the Ti Vector System

Tobacco plants were transformed by the leaf disc method utilizingAgrobacterium tumefaciens containing either plasmid pHB101 or pHB102 andthen the kanamycin resistant transformed tobacco plants wereregenerated.

Leaf disc transformation was performed in accordance with the procedureof Horsch et al⁶. Tobacco seeds (Nicotiana tabacum L. cv Samsun) weresurface sterilized with twenty per cent (20%) household bleach (dilutedone to five from the bottle) for ten minutes and then washed five timeswith sterile water. The seeds were sown on sterile MSO⁶ medium in GA-7boxes (Magenta Corporation, Chicago Ill.). The seedlings were grownunder moderate light for four to six weeks, and leaf tissue was excisedwith a sterile scalpel and cut into five-tenths square centimeter (0.5cm²) pieces.

The A. tumefaciens containing pHB101 or pHB102 which had been grown inYEP (east extract-peptone broth) medium were diluted one to ten withMSO⁶ for tobacco leaf pieces. Leaf pieces were inoculated by immersionin the diluted transformed A. tumefaciens culture and cocultured onregeneration medium MS 104⁶ for two days at twenty-seven degrees Celsius(27□C). Leaf pieces were then washed with sterile water to remove thefree A. tumefaciens cells and placed on fresh MS selection medium whichcontained two hundred micrograms per milliliter (200 □g/ml) kanamycin toselect for transformed plant cells and two hundred micrograms permilliliter (200 □g/ml) cefotaxime to inhibit bacterial growth. Leafpieces were subcultured every two weeks on fresh MS selection mediumuntil shoots appeared at the cut edges. As shoots formed at the edge ofthe leaf pieces and grew large enough for manual manipulation, they wereexcised (usually at three to six weeks after cocultivation withtransformed A. tumefaciens) and transferred to a root-inducing medium,e.g. MS rooting medium containing one hundred micrograms per milliliterof kanamycin (100 □g/ml). As roots appeared, the plantlets were eitherallowed to continue to grow under sterile tissue culture conditions ortransferred to soil and allowed to grow in a controlled environmentchamber.

D. Analysis of RNA from Transformed Tobacco

The regenerated kanamycin-resistant pHB101 and pHB102 transformedtobacco plants were analyzed by hybridizing RNA samples with a³² Plabelled probe encompassing the HBsAg gene coding region.

Total RNA from the leaves of the p HB101 transformed tobacco plants wasisolated as described Approximately four tenths of a gram (0.4 g) ofyoung growing leaf tissue from a transformed plant was frozen in liquidnitrogen and ground to a powder with a cold mortar and pestle. Thepowder was resuspended in five milliliters (5 ml) of RNA extractionbuffer composed of two hundred millimolar (0.2M) Tris-HCI, pH 8.6; twohundred millimolar sodium chloride (0.2M NaCI); twenty millimolarethylenediaminetetraacetic acid (20 mM EDTA) and two percent sodiumdodecyl sulfate (2% SDS) and immediately extracted with five milliliters(5 ml) of phenol saturated with ten millimolar (10 mM) Tris-HCI, pH 8.0per one millimole ethylenediaminetetraacetic acid (1 mM EDTA), and fivemilliliters (5 ml) of chloroform. After centrifugation at three thousandtimes gravity (3,000×G) to separate the phases, the upper aqueous layerwas removed and made to three tenths molar (0.3M) potassium acetate, pH5.2. The nucleic acids in the extract were precipitated with two and ahalf (2.5) volumes of ethanol, pelleted at eight thousand times gravity(8,000×G), dried under reduced pressure, resuspended in one milliliter(1 ml) of water, and reprecipitated with the addition of one milliliter(1 ml) of six molar (6M) ammonium acetate and five milliliters (5 ml) ofethanol. The final pellet was dried and resuspended in two tenths of amilliliter (0.2 ml) of water, and the concentration of RNA estimated bymeasuring the absorbance of the samples at 260 nanometers (nm), assumingthat a solution of one milligram per milliliter (1 mg/ml) RNA has anabsorbance of twenty-five (25) units.

Five micrograms of each RNA sample was denatured by incubation forfifteen minutes at sixty-five degrees Celsius (65□C) in twentymillimolar (20 mM) MOPS (3-N-morpholino) propanesulfonic acid, pH 7.0;ten millimolar (10 mM) sodium acetate; one millimolarethylenediaminetetraacetic acid (1 mM EDTA); six and one half percent(6.5% w/v) formaldehyde; fifty percent (50% v/v) formamide, and thenfractionated by electrophoresis in one percent (1%) agarose gels. Thenucleic acids were transferred to a nylon membrane by capillaryblotting⁵⁹ for sixteen hours in twenty-five millimolar (25 mM) sodiumphosphate, pH 6.5. Then the nucleic acids were crosslinked to themembrane by irradiation with ultraviolet (UV) light and the membranepretreated with hybridization buffer twenty-five hundredths molar(0.25M) sodium phosphate, pH 7.0; one millimolar ethylene diaminetetraacetic acid (1 mM EDTA); seven percent (7%) sodium dodecyl sulfate(SDS)! for one hour at sixty-eight degrees Celsius (68□C). The membranewas probed with 10⁶ counts per minute per milliliter (cpm/ml) ³²P-labelled random-primed DNA using a 700 base pair (bp) Bam HI-Acc Ifragment from plasmid pKS-HBS which includes most of the coding regionfor HBsAg. Blots were hybridized at sixty-eight degrees Celsius (68□C)in hybridization buffer and washed twice for five hundred and fifteenminutes with forty millimolar (40 mM) sodium phosphate, pH 7.0 per onemillimolar ethylene diaminetetraacetic acid (1 mM EDTA) per five percentsodium dodecyl sulfate (5% SDS) at sixty-eight degrees Celsius (68□C)and exposed to X-OMAT AR film for twenty hours.

The results of the RNA hybridization probe with selected transformantsharboring the plasmid pHB101construct and with a wild-type control (wt)can be seen in FIG. 6A. The signals were highly variable betweentransformants, as expected due to the effects of position of insertioninto the genomic DNA and differing copy number. The transcripts wereabout 1.2 kb in length by comparison with the RNA standards, which wasconsistent with the expected size. The wild-type control leaf RNA showedno detectable signal at this stringency of hybridization. Substantialsteady-state levels of MRNA which specifically hybridized with the HBsAgprobe was present in the leaves of selected transformants whichindicated that mRNA stability was not a problem for the expression ofHBsAg in tobacco leaves.

E. Analysis of Protein from Transformed Tobacco Plants

Protein was extracted from transformed tobacco leaf tissues byhomogenization with a Ten-Broek ground glass homogenizer (clearance 0.15mm) in five volumes of buffer containing twenty millimolar (20 mM)sodium phosphate, pH 7.0, one hundred fifty millimolar (150 mM) sodiumchloride, twenty millimolar (20 mM) sodium ascorbate, one-tenth percent(0.1%) Triton X-100, and five tenths millimolar (0.5 mM) PMSF, at fourdegrees Celsius (4□C). The homogenate was centrifuged at one thousandtimes gravity (1000×G) for five minutes and the supernatant centrifugedat twenty-seven thousand times gravity (27,000×G) for fifteen minutes.The 27,000×G supernatant was then centrifuged at one hundred thousandtimes gravity (100,000×G) for one hour and the pellet resuspended inextraction buffer. The protein in the different fractions was measuredby the Coomassie dye-binding assay (Bio-Rad). HBsAg protein was assayedby the AUSZYME Monoclonal kit (Abbott Laboratories, Abbott Park, Ill.)using the positive control, HBsAg derived from human serum, as thestandard. The positive control was diluted to give HBsAg protein levelsof nine hundredths to one and eight tenths nanograms (0.09-1.8 ng) perassay. After color development, the absorbance at four hundredninety-two nanometers (492 nm) was read and a linear relationship wasfound. As seen in FIG. 6B, the weld-type control plant contained nodetectable HBsAg protein (Column 1); fairly low levels of HBsAg proteinwere observed, ranging from three to ten nanograms per milligram (3-10ng/mg) soluble protein for the pHB101 construct (Columns 2 through 6);and from twenty-five to sixty-five nanograms per milligram (25-65 ng/mg)for the pHB102 construct (Columns 7 through 9). The reaction wasspecific because the wild-type tobacco showed no detectable HBsAgprotein. HBsAg from human serum and recombinant HBsAg (rHBsAg) fromplasmid-transformed yeast occur as approximately twenty nanometer (20nm) spherical particles consisting of protein embedded in a phospholipidbilayer. Ninety-five percent of the rHBsAg in the 27,000×G supernatantsof transgenic tobacco leaf extracts pelleted at 2000,000×G for thirtyminutes. This suggested a particle form. Thus, evidence was sought toascertain if rHBsAg in tobacco existed as particles.

F. Immunoaffinity Purification of HBsAg from Transformed Tobacco Plants

Transformed tobacco leaf extracts were tested for the presence ofmaterial which reacts specifically with monoclonal antibody toserum-derived HBsAg. Further tests were conducted to determine if therecombinant HBsAg material in the transformed tobacco leaves was presentas particles and the size range of the particles.

Monoclonal antibody against HBsAg, clone ZMHB1, was obtained from ZymedLaboratories (South San Francisco, Calif.). The immunogen source forthis antibody is human serum. The monoclonal antibody was bound toAffi-Gel HZ hydrazide gel (Bio-Rad Laboratories, Richmond, Calif.)according to the instruction supplied in the kit. The 100,000×Gresuspended soluble material was made to five tenths molar (0.5M) sodiumchloride and mixed with the immobilized antibody-gel by end-over-endmixing for sixteen hours at four degrees Celsius (4□C). The gel waswashed with ten volumes of PBS.5 ten millimolar (10 mM) sodiumphosphate, pH 7.0, five tenths molar (0.5M) sodium chloride! and tenvolumes of PBS.15 fifteen hundredths molar (0.15M) sodium chloride! andbound BBsAg eluted with two tenths molar (0.2M) glycine, pH 2.5. Theeluate was immediately neutral with Tris-base, and particles pelleted atone hundred and nine thousand times gravity (109,000×G) for one and ahalf hours at five degrees Celsius (5□C). The pelleted material wasnegatively stained with phosphotungstic acid and visualized withtransmission electron microscopy using a Phillips CMIO microscope. Thepresence of rHBsAg particles were revealed by negative staining andelectron microscopy, FIG. 7. rHBsAg particles ranged in diameter betweenten and forty nanometers (10-40nm). Most particles were between sixteenand twenty-eight nanometers (16-28 nm). These are very similar to theparticles observed in human serum,⁶⁹ although no rods were observed. TherHBsAg particles from yeast occur in a range of sizes with a mean ofseventeen nanometers (17 nm).² Thus rHBsAg produced in transgenictobacco leaves has a similar physical form to the human HBsAg.

G. Sucrose and Cesium Chloride Gradient Analysis of HBsAg fromTransgenic Tobacco

Further evidence of the particle behavior of rHBsAg was obtained fromsedimentation and buoyant density studies of the transgenic tobacco leafextracts.

Extras of the transgenic tobacco leaf tissue were made as described inthe protein analysis section and five tenths milliliter (0.5 ml) of the27,000×G supernatants were layered on linear eleven milliliter (11 ml)five to thirty percent (5-30%) sucrose gradients made in ten millimolar(10 mM) sodium phosphate, pH 7.0, fifteen hundredths molar (0.15M)sodium chloride or discontinuous twelve milliliters (12 ml) one and onetenth to one and four tenth grams per milliliter (1.1-1.4 g/ml) cesiumchloride gradients made in ten millimolar (10 mM) sodium phosphate, pH7.0 three milliliters (3 ml) each of one and one tenth, one and twotenths, one and three tenths, and one and four tenths grams permilliliter (1.1, 1.2, 1.3 and 1.4 g/ml) cesium chloride!. Positivecontrol HBsAg from the AUSZYME kit was also layered on separategradients. The sucrose gradients were centrifuged in a Beckman SW41Tirotor at thirty-three thousand revolutions per minute (33,000 rpm) forfive hours at five degrees Celsius (5□C), and fractionated into onemilliliter (1 ml) fractions while monitoring the absorbance at twohundred and eighty nanometers (280 nm). The cesium chloride gradientswere centrifuged in a Beckman SW40Ti rotor at thirty thousandrevolutions per minute (30,000 rpm) for twenty five hours at fivedegrees Celsius (5□C), and fractionated into five tenths milliliter (0.5ml) fractions. HBsAg in the gradient was assayed using the AUSZYME kitas described above.

FIG. 8 shows a sucrose gradient profile of rHBsAg activity from thetransgenic tobacco leaves harboring the plasmid construct pHB102. Thetransgenic tobacco rHBsAg sedimented with a peak near the 60S ribosomalsubunit, and the serum-derived HBsAg material sedimented in a somewhatsharper peak just slightly slower. This data is consistent with thefinding that human HBsAg sediments at 55S.⁷⁰ The observation that theplant rHBsAg material sedimented slightly faster and with a broader peakthan the human HBsAg is consistent with the larger mean size of therHBsAg plant particles and the wider range of particle sizes.

The buoyant density of the rHBsAg particles from transgenic tobaccoplants in cesium chloride, FIG. 9, was found to be approximately one andsixteen hundredths grams per milliliter (1.16 g/ml), while the humanHBsAg particles showed a density of about one and two tenths grams permilliliter (1.20 g/ml). Thus, the rHBsAg from the transgenic tobaccoplants exhibits sedimentation and density properties that are verysimilar to the subviral HBsAg particles obtained from human serum. Mostimportantly, HBsAg in the particle form is much more immunogenic thanthat found in the peptide form alone.²

H. Reproduction of HBsAg Transgenic Tobacco Plants

Reproduction of transgenic plants was accomplished as stated in ExampleI.

EXAMPLE III

A. Transformation of Tomato with HBsAg Gene

Tomato, Lycopersicom esculentum var. VFN8, was transformed as in ExampleH. B and C by the leaf disc method using Agrobacterium tumefaciensstrain LBA4404 as a vector, McComck et al., 1986.²³ A. tumefaciens cellsharboring plasmid pHB102, constructed as in Example II. A.2, whichcarries the HBsAg coding region fused to the tobacco etch virusuntranslated leader, Carrington & Freed, 1990,⁷³ and the cauliflowermosaic virus 35S promoter, were used to infect cotyledon explants fromseven day old seedlings. The explants were not preconditioned on feederplates, but infected directly upon cutting, and co-cultivated in theabsence of selection for two days. Explants were then transferred tomedium B, McCormick et al., 1986,²³ containing five-tenths milligramsper milliliter (0.5 mg/ml) carbenicillin and one-tenth milligram permilliliter (0.1 mg/ml) kanamycin for selection of transformed callus.Shoots were rooted in MS medium containing one-tenth milligram permilliliter (0.1 mg/ml) kanamycin but lacking hormones, and transplantedto soil and grown in a greenhouse.

Several independent kanamycin-resistant callus lines were obtained afterAgrobacterium-mediated transformation of the tomato variety VFN8. One ofthese lines regenerated shoots with high frequency and was rooted andgrown in soil in the greenhouse. The tissues from these plants were usedfor the protein and RNA analyses.

B. Quantitation of HBsAg in Leaves and Fruits

Plants tissues were extracted by grinding in a mortar and pestle withsolid carbon dioxide (CO₂), and suspended in three volumes of buffercontaining twenty millimolar (20 mM) sodium phosphate, one hundred fiftymillimolar sodium chloride (150 mM NaCl), five tenths millimolarphenylmethylsulfonyl fluoride (0.5 mM PMSF), one tenth percent (0.1%)Triton X-100, pH 7.0. After centrifuging the homogenate at ten thousandstimes gravity (10,000×g) for five minutes at four degrees Celsius (4□C),aliquots of the supernatant were assayed for total soluble protein bythe method of Bradford⁷⁴ and for HBsAg with the Auszyme II kit (AbbottLaboratories) as described in Example II. E.

HBsAg Levels in Transformed Tomato Tissues

In order to test for accumulation of HBsAg protein in transgenic plants,extracts of leaf and fruit were made, which were used for HBsAg-specificELISA. A standard curve was obtained using authentic HBsAg which wasderived from the serum of infected individuals. Table 1 shows the levelsof accumulation of HBsAg in leaves and ripe fruit of transgenic plants.Young leaf and red fruit from greenhouse-grown transgenic tomato plantswere extracted and assayed for total soluble protein and HBsAg asdescribed above. Similar tissues from untransformed control tomatoplants showed very low background for HBsAg.

The level found in tomato leaves is similar to the highest level foundin leaves of transgenic tobacco by Mason et a., 1992⁷², and represents0.007% of the total soluble protein. The amount of HBsAg in ripe fruitwas somewhat lower, 0.0043%, or 87 ng/g fresh weight. Similar extractsof untransformed tomato leaves showed negligible amounts of anti-HBsAgreactive material, at least 50-fold lower than the transformed plants.

The level of expression in the tomato fruit, although somewhat lower ona total protein basis, represents a substantial proportion of the wholeplant accumulation of HBsAg because the fruit are much more dense thanthe leaves. A small tomato weighing one hundred grams would containapproximately nine micrograms (9 □g) of HBsAg.

                  TABLE 1    ______________________________________    HBsAg Levels in Transgenic Tomato Leaf Fruit                ng/mg total    Organ       soluble protein (%)                             ng/g fresh weight    ______________________________________    Leaf        70 (0.007%)    Fruit (red) 43 (0.0043%) 87    ______________________________________

C. RNA Extraction and Northern Blotting

RNA was extracted as described in Example II. D., except that thetissues were ground with solid carbon dioxide (CO₂) instead of liquidnitrogen (N₂). RNA was fractionated and blotted to nylon membranes(Boehringer-Mannheim), fixed by irradiation on a ultraviolettransilluminator for three minutes, and air dried. Total RNA on the blotwas visualized by staining with twenty-five hundredths percent (0.25%)methylene blue per twenty-five hundredths molar sodium acetate (0.25MNaOAc), pH 4.5 for five minutes and destaining with water. The blot wasthen prehybridized in twenty-five hundredths molar (0.25M) sodiumphosphate, pH 7.0, ten millimolar ethylenediaminetetraacetic acid (10 mMEDTA), seven percent sodium dodecyl sulfide (7% SDS) for one hour atsixty-eight degrees Celsius (68□C) and probed with digoxygenin-labeledrandom-primed DNA made using the HBsAg coding region as templateaccording to the manufacturer's instructions (Genius 2 Kit,Boehringer-Mannheim). After washing the blot twice with forty millimolar(40 mM) sodium phosphate, pH 7.0, five percent sodium dodecyl sulfate(5% SDS) at sixty-eight degrees Celsius (68□C) and twice with fortymillimolar (40 mM) sodium phosphate, pH 7.0, one percent sodium dodecylsulfate (1% SDS) at sixty-eight degrees Celsius (68□C), the hybridizedRNA was detected by probing with anti-digoxygenin-alkaline phosphataseconjugate and developing color for sixteen hours according to themanufacturer's instructions (Genius 2 Kit, Boehringer-Mannheim).

The activity of the HBsAg gene in transgenic plants was assessed by RNAblotting. Total RNA isolated from transformed tomato leaves and greenfruit and from untransformed leaves was fractionated in a denaturingagarose gel, transferred to a nylon membrane, and hybridized withrandom-primed digoxygenin-labeled probe made using the HBsAg codingsequence as template. FIG. 10A shows that RNA from transformed tomatoleaf and fruit hybridized with the HBsAg probe, while RNA fromuntransformed leaf showed no detectable signal. The level of HBsAg mRNAin leaves was approximately three to five times greater than in fruit,on a total RNA basis. FIG. 10B shows a similar RNA blot stained withmethylene blue to reveal the total RNA pattern, and indicates that thesamples were loaded with equivalent amounts of total RNA. Thus, theHBsAg transgene is transcribed faithfully in transgenic tomato leaf andfruit, and accumulates to substantial levels. The yield of RNA from ripefruit was poor, and was not analyzed by RNA blotting.

D. Tissue Blotting for HBsAg Detection

Leaves of transformed or untransformed tomato plants were excised andpressed on fine-grain sandpaper before blotting abaxial side down onnitrocellulose. Tomato fruits were sectioned with a razor blade andpressed onto nitrocellulose for 30 sec. The blot was blocked with 5%nonfat dry milk in 10 mM sodium phosphate, pH 7.2, 140 mM NaCl, 0.05%Tween-20, 0.05% NaN3 (PBST)for 2 hr at 37□C. The blot was probed withmouse monoclonal anti-HBsAg (Zymed Laboratories) at 1:1000 dilution in2% nonfat dry milk in PBST for 2 hr at 23□C., before washing anddetection with goat anti-mouse IgG-alkaline phosphatase conjugate(BioRad) and development with NBT and BCIP according to manufacturer'sinstructions (Genius 2 Kit, Boehringer-Mannheim).

Tissue blots on nitrocellulose, probed with monoclonal anti-HBsAg, asseen in FIG. 11, graphically demonstrate the presence of HBsAg in thetransformed tomato tissues. Because this antibody does not react withSDS-denatured HBsAg, it was not possible to detect HBsAg on westernblots of SDS-PAGE fractionated leaf proteins. FIG. 11 shows a tissueblot of transformed and untransformed tomato leaf and transformed tomatofruit. The faint color of the untransformed leaf blot on the left isfrom chlorophyll; very little purple staining was observed. Thetransformed leaf on the right and the transformed fruit at bottom showedpurple precipitate indicating specific binding of the anti-HBsAgantibody.

EXAMPLE IV

A. Construction of Transmissible Gastroenteritis Virus PlasmidExpression Vector

The Transmissible Gastroenteritis Virus (TEGV) coding sequence TGEVS-protein as described in Sanchez et al., 1992⁷⁵ was obtained from Dr.Lisa Welter (Ambico-West Los Angeles, Calif.) as a PCR product clonedinto plasmid pGEM-T (Promega Corp., Madison, Wis.). The 5' end wastruncated six base pairs (6 bp) upstream of the translation initiationsite by digestion with HincII. The 1.2 kilobase (kb) HincII/XhoIfragment was isolated and ligated into plasmid pBluescript KS(Stratagene, La Jolla, Calif.) which was previously digested with SmaIand XhoI. The resulting plasmid, pTG5', was then digested with BamHI andXhoI and the 1.2 kilobase (kb) fragment isolated. The 3.3 kilobase (kb)Xhol/SstI fragment, representing the 3' end of the S-protein proteincoding region, was isolated and ligated together with the 1.2 kilobase(kb) BamHI/Xhol fragment from plasmid pTG5', representing the 5' end ofthe S-protein coding region, into plasmid pBluescript KS that had beendigested with BamHI and SstI. The resulting plasmid, pKS-TG, was thendigested with BamHI and SstI to give the entire 4.5 kilobase (kb)S-protein coding sequence, which was then ligated into the potato tuberexpression vector plasmid pPS20⁷⁶ that was digested with BamHI and SstIand isolated from the GUS coding region. Plasmid pPS20 is a derivativeof pBI101⁷⁷, and contains a kanamycin resistance cassette for selectionof transformed plants. The resulting plasmid, pPS-TG, contains theS-protein coding region downstream of the patatin promoter, which drivestuber-specific expression in potato plants, and followed by the nopalinesynthase polyadenylation signal.

B. Potato Transformation

Agrobacterium tumefaciens LBA4404 was transformed with plasmid pPS-TG bythe freeze-thaw method of An⁷⁸, and the plasmid structure verified byrestriction digestion. The Agrobacterium stain harboring plasmid pPS-TGwas used for transformation of the potato variety "Atlantic." The potatotransformation protocol was as described in Wenzler⁷⁹ and shoots wereregenerated on media containing fifty milligrams per liter (50 mg/L)kanamycin. Microtubers were induced on nodal stem segments as describedby Wenzler.⁷⁹

C. Analysis of S-protein Expression in Microtubers

Total RNA was extracted from microtubers using the method of Mason andMullet⁸⁰ , except that the microtubers were homogenized in three volumesof buffer in microcentrifuge tubes with pellet pestles, rather thangrinding with liquid nitrogen (N₂). The RNA samples were assayed forS-protein mRNA by RNA dot blotting⁸¹ and hybridization with adigoxygenin-labeled probe made by random-primed DNA synthesis (Genius 2Kit, Boehringer-Mannheim, Indianapolis, Ind.). The 2.2 kilobase (kb)XhoI/XbaI fragment from the coding region of the TGEV S-protein gene wasthe template for probe synthesis. Hybridization and detection were doneas per kit instructions (Genius 2 Kit, Boehringer-Mannheim,Indianapolis, Ind.), except that the hybridization buffer containedtwenty-five hundredths molar (0.25M) sodium phosphate, pH 7.0, fivepercent (5%) sodium lauryl sulfate, and ten millimolarethylenediaminetetraacetic acid (10 mM EDTA). The results were onlyqualitative, but indicate that there was a range of different levels ofexpression of S-protein MRNA among the independent transformants, as isexpected for a random insertion of the foreign gene into the host plantgenome.

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The foregoing description of the invention has been directed to aparticular preferred embodiments in accordance with the requirements ofthe patent and statutes and for purposes of explanation andillustration. It will become apparent to those skilled in the art thatmodifications and changes may be made without departing from the scopeand the spirit of the invention.

We claim:
 1. A food comprising transgenic plant material capable ofbeing ingested for its nutritional value, said transgenic plantexpressing a recombinant immunogen derived from Hepatitis virus.
 2. Thefood of claim 1 wherein said immunogen is Hepatitis B surface antigen.3. The food of claim 1 wherein said plant is selected from the groupconsisting of: tomato and potato.
 4. A food comprising transgenic plantmaterial capable of being ingested for its nutritional value, saidtransgenic plant expressing a recombinant immunogen derived fromTransmissible Gastroenteritis Virus.
 5. The food of claim 3 wherein saidimmunogen is Transmissible Gastroenteritis Virus S.
 6. The food of claim4 wherein said plant is selected from the group consisting of: tomatoand potato.
 7. The food of claim 1 or 3 wherein said transgenic plantmaterial is selected from the group consisting of: edible fruit, leaves,juices, roots, and seed of said plant.